TGF-β1 was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Curcumin and rabbit monoclonal anti-α-SMA antibody (SP171) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit polyclonal anti-collagen type I (ColI; ab34710) and -von Willebrand factor (ab6994) primary antibodies were purchased from Abcam (Cambridge, MA, USA), whereas mouse monoclonal anti-vimentin antibody (zm-0260), -glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (TA-08), and horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse immunoglobulin (Ig)G secondary antibodies (ZB-2301 and ZB-2305) were purchased from Zhongsan Jinqiao Biotechnology Co., Ltd. (Beijing, China). Phosphorylated and non-phosphorylated monoclonal rabbit anti-rat Smad2 (#3108 and #5339) and p38 (#9215 and #2371) primary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). SB431542, a TGF-βR-Smad2/3 inhibitor was purchased from Cayman Chemical Company (Ann Arbor, MI, USA) and SB20358, a p38 inhibitor, was purchased from Merck Millipore (Darmstadt, Germany). Cell culture materials and TRIzol® reagent were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The DNase and primers used for the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were purchased from Sunbiotech (Beijing, China).

The present study was conducted with the approval of the Ethics Committee of Experimental Animal Center of Shanxi Cardiovascular Hospital (Taiyuan, China), according to the regulations outlined by the National Institutes of Health Guidelines on the Use of Laboratory Animals. CFs were isolated from neonatal (1–3-days-old) Sprague-Dawley rats via trypsin digestion methods, as previously described (18). CFs were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, and were passaged using trypsin (1:3). Second passage CFs were used in the present study and were serum-starved for 24 h at 80% confluence in order to induce quiescence. Immunocytochemical analysis demonstrated that the purity of the CFs used was >95%, according to positive staining for vimentin and negative staining for von Willebrand factor.

In order to elucidate the potential effects of curcumin on the various signaling pathways in TGF-β1-induced CFs, CFs were pretreated with 20 µmol/l curcumin, 10 µM TGF-βR-Smad2 inhibitor (SB431542) or 10 µM p38 MAPK inhibitor (SB203580) for 30 min, prior to treatment with recombinant 10 ng/ml TGF-β1 for 24 h. In order to investigate the activation of Smad2 and p38, CFs were treated with 10 ng/ml TGF-β1 for 1 h. Cells were subsequently harvested and stored at −80°C prior to the determination of protein and mRNA expression levels.

CFs were cultured on coverslips in 6-well plates (2.5×10⁵ cells/well). Growth was arrested and CFs were treated as described earlier. Following a 24-h treatment, CFs were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 (Applygen Technologies, Inc., Beijing, China). Non-specific binding was blocked via incubation with 10% normal goat serum (Applygen Technologies, Inc.). Subsequently, α-SMA was detected using a Cy5-conjugated goat anti-rabbit α-SMA polyclonal antibody (Abcam; ab6564) and stained cells were visualized using the BX51 Fluorescence Microscope (Olympus Corporation, Tokyo, Japan).

CFs were harvested in lysis buffer (Applygen Technologies, Inc.) containing 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, 1 mM Na₃VO₄ (pH 7.5), 1 mM phenylmethanesulfonyl fluoride, 1 mM benzamidine, 10 µg/ml leupeptin and 10 µg/ml aprotinin. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA), according to the manufacturer's protocol. Following boiling for 1 min to denature, 20 µg protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Membranes were blocked with 5% fat-free milk in Tris-buffered saline with Tween-20 (TBST; 10 mM Tris HCl, 150 mM NaCl and 0.1% Tween-20) for 1 h at room temperature, and incubated overnight with primary antibodies (1:2,500) at 4°C. Following each incubation, membranes were washed three times for 10 min with TBST and subsequently incubated with HRP-conjugated secondary antibody (1:5,000) for 1 h at room temperature. Using chemiluminescence (Applygen Technologies, Inc.), the membranes were scanned and quantified using Quantity-One software, version 4.2 (Bio-Rad Laboratories, Inc.), and the results were presented as the optical density of phosphorylated-protein/total protein or of the target protein/GAPDH.

Total RNA was extracted from CFs using TRIzol® reagent and treated with DNase, according to the manufacturer's protocols. RT-qPCR analyses were performed using an ABI 7300 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). For each sample, 1 µg RNA was utilized to synthesize cDNA using the Reverse Transcriptase kit (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol. The specific primer sequences used were as follows: α-SMA forward, 5′-CATCAGGAACCTCGAGAAGC-3′, and reverse, 5′-TCGGATACTTCAGGGTCAGG-3′; ColI forward, 5′-CATAAAGGGTCATCGTGGCTTC-3′, and reverse, 5′-GTGATAGGTGATGTTCTGGGAG-3′; and GAPDH forward, 5′-AACCTGCCAAGTATGATGACATCA, and reverse, 5′-TTCCACTGATATCCCAGCTGCT-3′. Target genes were amplified using SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR cycling conditions were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 35 sec and 60°C for 1 min, with a final extension step of 72°C for 5 min. Relative mRNA levels were calculated using the 2^−ΔΔCq method (19), where ΔCq was the difference between GAPDH and target gene critical threshold cycle (Cq) values.

Statistical analyses in this study were conducted using the SPSS software, version 16.0 (SPSS, Inc., Chicago, IL, USA). The results are presented as the mean ± standard error of the mean. Between-groups differences were assessed using one-way factorial analysis of variance. P<0.05 was considered to indicate a statistically significant difference. Each assay in the present study was performed in triplicate.

To investigate the effects of curcumin on the differentiation of CFs into myofibroblasts, western blotting was used to detect the protein expression levels of α-SMA and ColI (Fig. 1A). The results demonstrated that curcumin significantly suppressed the TGF-β1-induced protein expression of α-SMA and ColI in CFs (P<0.05), in a dose-dependent manner. These anti-differentiation effects were further validated by immunofluorescence staining of α-SMA (Fig. 1B). As compared with untreated CFs, cells induced by 10 ng/ml TGF-β1 exhibited bright fluorescence staining signals for α-SMA and prominent stress fibers; by contrast, treatment with 20 µM curcumin significantly attenuated α-SMA fluorescence signals and morphological characteristics of CFs induced by 10 ng/ml TGF-β1 (P<0.05). Furthermore, RT-qPCR was performed to analyze the mRNA expression levels of α-SMA and ColI. The results demonstrated that α-SMA and ColI mRNA expression levels in TGF-β1-induced CFs were also significantly and dose-dependently decreased following treatment with curcumin (P<0.05; Fig. 2).

To investigate the downstream signaling pathways in TGF-β1-stimulated CFs, the levels of activated (phosphorylated) Smad2 and p38 were measured at various time points between 15 and 60 min after TGF-β1 administration (Fig. 3). As determined by western blotting, phosphorylated Smad2 and p38 (p-Smad2 and p-p38) levels were significantly increased following stimulation with TGF-β1 (P<0.05). Total Smad2 and p38 protein expression levels were also examined and found to be unaffected by TGF-β1 administration. These results indicate that treatment with TGF-β1 activates the Smad2 and p38 signaling pathways in CFs.

To assess whether Smad2 and p38 were associated with the TGF-β1-induced differentiation of CFs, the Smad2 and p38 inhibitors, SB431542 and SB203580, respectively, were used to block these signaling pathways (Fig. 4). When administered alone, SB431542 and SB203580 exhibited no significant effect on cardiac fibroblast differentiation, as detected by consistent mRNA α-SMA and ColI expression levels (P>0.05). However, pretreatment with SB431542 or SB203580 prior to TGF-β1 administration significantly suppressed the TGF-β1-induced mRNA expression levels of α-SMA and ColI (P<0.05).

In CFs, TGF-β1 administration induced an early and significant increase in p-Smad2 expression levels over the baseline value in 30 min, as analyzed using western blotting (P<0.05; Fig. 5). Pre-treatment with curcumin induced a significant decrease in the degree of Smad2 phosphorylation and, as hypothesized, SB431542 (P<0.05), which is a well established TGF-βR-Smad2 inhibitor, effectively eliminated the activation of Smad2. Furthermore, pre-treatment with the p38 inhibitor, SB203580, also significantly attenuated the TGF-β1-induced Smad2 phosphorylation (P<0.05).

The effects of curcumin on the TGF-β1-induced activation of the p38 signaling pathway were also examined. Western blot analysis demonstrated that, in TGF-β1-stimulated CFs, curcumin pre-treatment significantly decreased p-p38 expression levels, and SB203580 effectively eliminated p38 activation (P<0.05). Furthermore, SB431542 administration also significantly inhibited the phosphorylation of p38, thus preventing its activation (P<0.05; Fig. 6).